Pneumocystis carinii (P. jiroveci in human disease) causes a severe pneumonia in irnmunocornprornised individuals, such as those with AIDS. The number of alveolar macrophages is decreased in humans with Pneumocystis pneumonia (Pep). In rat and mouse Pep models, alveolar macrophage number is decreased by approximately 60% mainly due to the increased rate of apoptosis in alveolar macrophages. During Pep, polyamine (spermidine, acetylspermine, and acetylspermidine) levels are greatly increased in the alveoli and alveolar macrophages. Bronchoalveolar lavage (BAL) fluids from animals with Pep are able to induce apoptosis in normal alveolar macrophages, and depletion of polyamines from these BAL fluids abrogates their ability to induce apoptosis. This proposal will use steroid-immunosuppressed rats and L3T4 cell- depleted mice to test the hypothesis that alveolar macrophages apoptosis is caused directly by polyamines or indirectly by reactive oxygen species that are generated as the result of polyamine catabolism during Pep. Experiments will be performed to determine whether the polyamines present in the alveoli and alveolar macrophage during Pep are derived from Pneumocystis organisms, alveolar macrophages, and/or lung epithelial cells. The levels of each specific polyamine needed to induce apoptosis in alveolar macrophages will be determined. Changes in polyamine uptake by alveolar macrophages will also be assessed. The relationship between increased levels of reactive oxygen species and polyamines will be investigated. Pro- and anti-apoptosisfactors that are involved in the apoptosis will be identified, and effects of polyamines on the expression and the activity of these factors will be investigated. The involvement of extrinsic and intrinsic (mitochondrial) apoptosis pathways will be studied by identifying factors that alter mitochondrial membrane potential leading to release of cytochrome c to the cytoplasm and the contribution of death receptors and their ligands to the apoptosis. Effects of down regulation of anti-apoptotic factors on the resistance of alveolar macrophage to polyamine-mediated, Pneumocystis-induced apoptosis will also be investigated. Since preliminary studies in both rats and mice with Pep indicate that inhibition of apoptosis in alveolar macrophages ameliorates disease progression or even resolves the infection, the proposed studies may lead to new treatments for Pneumocystis pneumonia.